KR20220070275A - Oligonucleotide-based ex vivo cell therapy - Google Patents

Abstract

The present invention provides a method of reducing the expression of a target RNA in an isolated cell in preparation for cell therapy, comprising incubating an isolated cell comprising the target RNA with an antisense oligonucleotide without the use of transfection means, , wherein the antisense oligonucleotide is administered at least once to the isolated cells within a period of 0 to 21 days, wherein the antisense oligonucleotide hybridizes with the target RNA from day 0 to up to 8 weeks of incubation with the antisense oligonucleotide to the target RNA It relates to a method for reducing the transcription of or reducing the expression of a protein encoded by a target RNA or a combination thereof. The present invention also relates to an isolated cell obtainable by the method of the present invention and a pharmaceutical composition comprising the isolated cell. The isolated cells and pharmaceutical compositions are used in methods of preventing and/or treating diseases.

Description

Oligonucleotide-based ex vivo cell therapy

The present invention relates to an ex vivo method for reducing target RNA in an isolated cell in preparation for use in cell therapy, the isolated cell obtainable via the method, and a pharmaceutical composition comprising the isolated cell. The isolated cells and pharmaceutical compositions are for use in a method of preventing and/or treating a disease.

Cell therapy (also called cellular therapy or cytotherapy) is the injection, implantation, or insertion of a cellular material into a patient, i.e., the injection, implantation, or insertion of a cellular material into living intact cells. It is therapy. The cells may be from a patient (autologous cells) or a donor (allogeneic cells). Cells used in cell therapy can be classified according to their potential to transform into different cell types. Pluripotent cells can transform into any cell type in the body and multipotent cells can transform into other cell types, but their repertoire is more limited than that of pluripotent cells. Differentiated or primary cells are of the fixed type. The type of cells administered is dictated by the treatment. For example, T cells capable of fighting cancer cells via cell-mediated immunity can be injected during an immunotherapy course. Cell therapy targets many clinical indications in multiple organs and by different modes of cell delivery. Thus, the specific mechanisms of action involved in therapy are broad.

For example, stem or progenitor cell engraftment, differentiation and long-term replacement of damaged tissue. In this paradigm, pluripotent or unipotent cells differentiate into specific cell types either in the laboratory or after reaching the site of injury (via local or systemic administration). These cells then integrate at the site of damage to replace the damaged tissue and thus promote improvement in the function of the organ or tissue. An example of this is the use of cells to replace myocardial cells after myocardial infarction.

Cells with the ability to release soluble factors such as cytokines, chemokines and growth factors that act in a paracrine or endocrine manner. These factors promote self-healing of an organ or region. Cells delivered (via local or systemic administration) survive for a relatively short period of time (days to weeks) and then die. This includes cells that have undergone epigenetic changes or genetic engineering that naturally secrete the relevant therapeutic factors or cause the cells to release specific molecules in large quantities. Examples of this include cells that secrete factors that promote angiogenesis, anti-inflammatory and anti-apoptosis.

Cells such as immune cells, blood cells or skin cells are generally capable of regenerating ex vivo given the correct conditions. This allows differentiated adult immune cells to be used for cell therapy. Cells can be removed from the body, isolated from the mixed cell population, transformed, and then expanded before returning to the body. A recently developed cell therapy involves the delivery of adult self-renewing T lymphocytes that have been genetically modified to increase immune potency to kill disease-causing cells.

Potential applications of cell therapy include the treatment of cancer, autoimmune diseases, urinary problems and infectious diseases, reconstruction of damaged cartilage in joints, repair of spinal cord damage, improvement of a weakened immune system, or support of patients with neurological disorders.

The disadvantage is unsatisfactory activity, and thus the different cell therapies and the unsatisfactory results of the cell therapies often lead to serious side effects, some of which are life-threatening, which must be managed by experienced professionals. These side effects occur, for example, when the altered cells proliferate rapidly and release substances called cytokines in large quantities. Severe cytokine release syndrome can cause life-threatening multi-organ damage and brain swelling. Thus, modified cells used in cell therapy exhibit the ability to induce expected and unexpected toxicities, including cytokine release syndrome, neurological toxicity, "on target/off tumor" recognition and anaphylaxis. have it

Therefore, there is an urgent need to develop cell therapies with reduced side effects and at least the effectiveness or even increased effectiveness of existing cell therapies. The present invention satisfies these needs.

The present invention provides a method for reducing the expression of a target RNA in an isolated cell in preparation for cell therapy, comprising incubating an isolated cell comprising the target RNA with an antisense oligonucleotide without the use of transfection means, wherein the antisense oligonucleotide is administered at least once to the isolated cells within a period of 0 to 21 days, wherein the antisense oligonucleotide hybridizes with the target RNA from day 0 to up to 8 weeks of incubation with the antisense oligonucleotide, and It relates to a method of reducing transcription or reducing transcription of a target RNA or reducing expression of a protein encoded by a target RNA or a combination thereof. The target RNA is, for example, selected from the group consisting of mRNA, pre-mRNA, lncRNA and/or miRNA.

A cell of the invention is, for example, an immune cell. Immune cells consist of, for example, T cells, dendritic cells, natural killer (NK) cells, peripheral blood mononuclear cells (PBMC), stem cells such as hematopoietic stem cells and/or induced pluripotent stem cells, B cells and combinations thereof. selected from the group. Alternatively or additionally, the immune cell is a T-cell and the target RNA is for example PD-1, TIGIT, TIM-3, LAG-3, TGFBR, CD39, CD73, 2B4, BTLA, VISTA, CD304, PQR-prot , Chop, XBP1, PERK, FOXP3, GMCSF, IFNg, TNFa, TGFb, IL-1, IL-2, IL-6, IL-10, IL-12, IL-17, IL-9, STAT3, IL-6 or a target RNA encoding a protein that affects the efficacy and/or safety of immune cells, selected from the group consisting of receptors and combinations thereof, for example BID, BIM, BAD, NOXA, PUMA, BAX, BAK, BOK, It encodes a protein that affects proliferation and/or survival of immune cells, selected from the group consisting of BCL-rambo, BCL-Xs, Hrk, Blk, BMf, p53, and combinations thereof.

Isolated cells are genetically modified, for example, by gene transfer techniques before or after incubating the cells with antisense oligonucleotides. Optionally, isolated genetically modified cells, such as immune cells, are expanded before or after incubating the cells with antisense oligonucleotides.

The method of reducing the expression of a target RNA in an isolated cell in preparation for use in cell therapy of the present invention comprises optionally purifying the isolated cell before and/or after incubating the cell, such as an immune cell, with an antisense oligonucleotide. additional steps are included.

Optionally, the method further comprises enriching an isolated cell, such as an immune cell, before and/or after incubating the immune cell with an antisense oligonucleotide, optionally wherein the antisense oligonucleotide is added to the isolated immune cell .

Optionally, the method comprises freezing isolated cells, such as immune cells, with antisense oligonucleotides, prior to incubation of immune cells with antisense oligonucleotides, incubating cells with antisense oligonucleotides, or a combination thereof. further comprising the step of preserving.

In the case of an isolated immune cell used in a method of reducing the expression of a target RNA in an isolated cell in preparation for use in cell therapy, the cell is a T-cell, or a dendritic cell, a natural killer (NK) cell, a peripheral blood mononuclear cells (PBMCs), stem cells such as hematopoietic stem cells and/or induced pluripotent stem cells, B cells, and combinations thereof.

Target RNA can be, for example, PD-1, TIGIT, TIM-3, LAG-3, TGFBR, CD39, CD73, 2B4, BTLA, VISTA, CD304, PQR-prot, Chop, XBP1, PERK, FOXP3, GMCSF, IFNg , TNFa, TGFb, IL-1, IL-2, IL-6, IL-10, IL-12, IL-17, IL-9, STAT3, selected from the group consisting of IL-6 receptors and combinations thereof, immune, Affect the efficacy and/or safety of cells such as dendritic cells, natural killer (NK) cells, peripheral blood mononuclear cells (PBMC), stem cells such as hematopoietic stem cells and/or induced pluripotent stem cells, B cells and combinations thereof or a target RNA that encodes a protein that affects It encodes a protein that affects the proliferation and/or survival of immune cells, selected from the group consisting of.

In the method of the present invention, the antisense oligonucleotide is for example 0 days to 21 days, 0 days to 20 days, 0 days to 19 days, 0 days to 18 days, 0 days to 17 days, 0 days to 16 days, 0 days 1 to 15 days, 0 to 14 days, 0 to 13 days, 0 to 12 days, 0 to 11 days, 0 to 10 days, 0 to 9 days, 0 to 8 days, 0 days to administered within a period of 7 days, 0-6 days, 0-5 days, 0-4 days, 0-3 days, 0-2 days or 0-1 days. The antisense oligonucleotide is administered, for example, every day, every 2 days, every 3 days, every 4 days, every 5 days, every 6 days, every 7 days, every 8 days, every 9 days or every 10 days of a period.

Optionally, in the methods of the invention, the antisense oligonucleotide hybridizes with two or more target RNAs administered, for example, at the same time point during the same time period or at different time points during the same or different time period.

The present invention further relates to a cell, such as an isolated immune cell, for use in a method of preventing and/or treating a disease, wherein the isolated immune cell is derived from a patient suffering from a disease or a healthy subject, and According to the method, incubation is performed with an antisense oligonucleotide that hybridizes with the target RNA to reduce expression of the target RNA, and after incubation of the isolated immune cells with the antisense oligonucleotide, the isolated immune cells are reintroduced into the patient or introduced into the patient. do. Accordingly, immune cells can be obtained by the method of the present invention and used in a method for preventing and/or treating a disease.

The present invention also relates to a pharmaceutical composition comprising the isolated immune cells of the present invention and a pharmaceutically acceptable excipient for use in a method for preventing and/or treating a disease. A patient suffering from a disease or a healthy subject is, for example, a human or non-human animal. The disease is, for example, selected from the group consisting of cancer, autoimmune disease, graft-versus-host disease, and combinations thereof.

All documents cited or referenced herein (“documents cited herein”) and all documents cited or referenced in documents cited herein refer to any product mentioned herein or in any document incorporated herein by reference. It is incorporated herein by reference, along with any manufacturer's instructions, descriptions, product specifications, and product specifications, and may be used in the practice of the present invention. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.

1 depicts robust knockdown of target RNA independent of the initial seeding density of isolated cells.
Figure 2 shows the reduction of target RNA expression after cryopreservation based on previous antisense oligonucleotide incubation.
3 depicts a parallel decrease in expression of different target RNAs through parallel incubation with two different antisense oligonucleotides in dendritic cells.
Figure 4a depicts a parallel decrease in expression of different target RNAs, and Figure 4b depicts a parallel decrease in expression of different proteins through parallel incubation with two different antisense oligonucleotides in T cells.
5 shows superior target RNA reduction of target-specific antisense oligonucleotides in T cells compared to target-specific siRNA.

The present invention relates to a method for reducing the expression of a target RNA in an isolated cell, such as an immune cell, in preparation for use in cell therapy. The method comprises incubating an isolated cell, such as an immune cell, comprising a target RNA, such as a gymnotic transfection, with an antisense oligonucleotide without the use of transfection means. The antisense oligonucleotide is administered at least once to an isolated cell, such as an immune cell, within a period of 0 to 21 days. Antisense oligonucleotides hybridize with the target RNA from day 0 to up to 8 weeks of incubation with the antisense oligonucleotide and reduce transcription of the target RNA or decrease the expression of a protein encoded by the target RNA or a combination thereof. Since administration of the antisense oligonucleotides of the present invention does not permanently block the transcription, function and/or expression of the target RNA, side effects based on permanent blockade of RNA transcription, function and/or expression are avoided. In addition, administration of antisense oligonucleotides in the absence of transfection means significantly reduces stress on cells and reduces or even avoids side effects caused by other transfection means.

Hereinafter, the elements of the present invention will be described in more detail. While these elements are listed together with specific embodiments, it should be understood that they may be combined in any manner and in any number to create additional embodiments. The variously described examples and embodiments should not be construed as limiting the invention to only the embodiments explicitly described. It is to be understood that this description supports and includes embodiments that combine the explicitly described embodiments with any number of the disclosed elements. Moreover, any permutations and combinations of all elements described in this application are to be considered disclosed by the description of this application unless the context dictates otherwise.

Throughout this specification and claims, unless the context requires otherwise, the word "comprise" and variations such as "comprises" and "comprising" refer to the member mentioned; The inclusion of an integer or step, or group of members, integers or steps, is not implied, but not the exclusion of any other member, integer or step, or group of members, integers or steps. Terms (“a”, “an” and “the” in the original text) and similar referents used in the context of describing the invention (especially in the context of the claims) are not otherwise indicated herein or are expressly disclaimed by context Unless otherwise stated, it will be construed to include both the singular and the plural. Recitation of a range of values herein is merely intended to serve as a shorthand method of referring individually to each individual value subsumed within that range. Unless otherwise indicated herein, each individual value is incorporated herein as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples or illustrative language (eg, “such as (eg)”, “for example”) provided herein is merely to better exemplify the invention, and the invention claimed does not otherwise limit the scope of No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

The cells of the invention are, for example, immune cells, stem cells, pluripotent stem cells, such as induced pluripotent stem cells, embryonic stem cells, skin stem cells, umbilical cord blood stem cells, mesenchymal stem cells, neural stem cells, or combinations thereof. Immune cells include, for example, T-cells, or dendritic cells, natural killer (NK) cells, peripheral blood mononuclear cells (PBMC), stem cells such as hematopoietic stem cells and/or induced pluripotent stem cells, B cells and their is selected from the group consisting of combinations.

When T-cells are selected, T-cells are for example PD-1, TIGIT, TIM-3, LAG-3, TGFBR, CD39, CD73, 2B4, BTLA, VISTA, CD304, PQR-prot, Chop, XBP1 , PERK, FOXP3, GMCSF, IFNg, TNFa, TGFb, IL-1, IL-2, IL-6, IL-10, IL-12, IL-17, IL-9, STAT3, IL-6 receptor, BID, Expresses one or more factors selected from the group consisting of BIM, BAD, NOXA, PUMA, BAX, BAK, BOK, BCL-rambo, BCL-Xs, Hrk, Blk, BMf, p53, and combinations thereof. When a T-cell is selected, the target RNA can be, for example, PD-1, TIGIT, TIM-3, LAG-3, TGFBR, CD39, CD73, 2B4, BTLA, VISTA, CD304, PQR-prot, Chop, XBP1, PERK, FOXP3, GMCSF, IFNg, TNFa, TGFb, IL-1, IL-2, IL-6, IL-10, IL-12, IL-17, IL-9, STAT3, IL-6 receptors and combinations thereof or encoding a protein that affects the efficacy and/or safety of T-cells, selected from the group consisting of, BID, BIM, BAD, NOXA, PUMA, BAX, BAK, BOK, BCL-rambo, BCL-Xs, Hrk, It encodes a protein that affects proliferation and/or survival of T-cells, selected from the group consisting of Blk, BMf, p53, and combinations thereof.

Immune cells such as T cells, dendritic cells, natural killer cells, peripheral blood mononuclear cells, stem cells such as hematopoietic stem cells and/or induced pluripotent stem cells, B cells are antigen-specific receptors such as, for example, chimeric antigen receptors. or genetically modified to express an immune cell receptor, such as a T cell receptor. These cells can exert antitumor functions by recognizing antigens on the surface of tumor cells through antigen-specific receptors and inducing activation of immune cells such as T cells. Activated immune cells, such as T cells, induce the destruction of tumor cells by, for example, releasing cytokines and toxic molecules.

Reducing the expression of a target RNA according to the present invention means reducing the transcription, function and/or expression of different amounts of the target RNA to maximally complete inhibition. The level of transcription, function and/or expression in a cell is determined, for example, by measuring and comparing the level of transcription, function and/or expression of the target RNA before and after treatment, ie, administration of the oligonucleotide.

The target RNA is, for example, mRNA, pre-mRNA, lncRNA and/or miRNA. The oligonucleotide hybridizes to a specific sequence of the target RNA and reduces the transcription, function and/or expression of the target RNA consisting of or comprising such sequence. The target RNA is directly or indirectly involved, for example, in the induction and/or maintenance of a disease. The target RNA is, for example, increased proliferation, reduced exhaustion, faster cell growth, increased metabolic activity, improved function, improved immunomodulatory effect and increased secretome efficiency and/or directly or indirectly affect the increase in potency. The methods of the invention result in a decrease in the amount of target RNA in a cell, such as an immune cell.

lncRNAs can function, for example, as signals, baits, scaffolds, guides, enhancer RNAs and even short peptides. The main function of signaling lncRNAs is, for example, to serve as molecular signaling that regulates transcription in response to various stimuli. Bait lncRNAs limit the availability of regulatory factors, for example, by presenting a "bait" binding site. lncRNAs regulate transcription by sequestering regulatory factors, including, for example, transcription factors, catalytic proteins, subunits of larger chromatin modification complexes, as well as miRNAs, thereby reducing their availability. Transcripts of the scaffold class of lncRNAs play a structural role by providing a platform for the assembly of multi-component complexes such as, for example, ribonucleoprotein (RNP) complexes. The guide lncRNA interacts with the RNP and directs the RNP, for example, to a specific target gene. These guide lncRNAs are, for example, essential for proper localization of RNPs. Enhancer RNA (eRNA) is produced from enhancer regions and affects, for example, the three-dimensional (3D) configuration of DNA known as "chromatin interactions". In addition, lncRNAs encode, for example, short regulatory peptides.

Target RNA can be for example PD-1, TIGIT, TIM-3, LAG-3, TGFBR, CD39, CD73, 2B4, BTLA, VISTA, CD304, PQR-prot, Chop, XBP1, PERK, FOXP3, GMCSF, IFNg, Immune cells selected from the group consisting of TNFa, TGFb, IL-1, IL-2, IL-6, IL-10, IL-12, IL-17, IL-9, STAT3, IL-6 receptors, and combinations thereof. encoding proteins that affect the efficacy and/or safety of, BID, BIM, BAD, NOXA, PUMA, BAX, BAK, BOK, BCL-rambo, BCL-Xs, Hrk, Blk, BMf, p53, and combinations thereof It encodes a protein that affects the proliferation and / or survival of immune cells, selected from the group consisting of.

Oligonucleotides have direct and/or indirect effects: factors of interest, eg PD-1, TIGIT, TIM-3, LAG-3, TGFBR, CD39, CD73, 2B4, BTLA, VISTA, CD304, PQR -prot, Chop, XBP1, PERK, FOXP3, GMCSF, IFNg, TNFa, TGFb, IL-1, IL-2, IL-6, IL-10, IL-12, IL-17, IL-9, STAT3, IL hybridizes to and/or ( direct effect), which hybridizes with the target RNA of another factor that affects the factor of interest, eg inhibits or activates the factor of interest (indirect effect).

The oligonucleotides of the invention are, for example, antisense oligonucleotides, siRNAs or aptamers. Oligonucleotides include, for example, cross-linked nucleotides, such as locked nucleic acids (LNAs, eg, 2',4'-LNAs), cET, ENA, 2' fluoro-modified nucleotides, 2'O - contain one or more modifications, such as methyl modified nucleotides, morpholinos, or combinations thereof.

Reduction of target RNA in isolated cells may result, for example, in higher proliferation of and in isolated cells, less exhaustion, faster growth, more potent secretome, higher metabolic activity, better function of and in isolated cells, respectively. , resulting in an improved immunomodulatory effect, fewer side effects, a favorable cytokine profile and/or higher potency.

The cells used in the methods of the invention are isolated, for example, from a human or non-human animal. A human animal is, for example, a human of any genetic background; Non-human animals include mammals of any genetic background, such as horses, cattle, pigs, sheep, cats, dogs, guinea pigs, hamsters, and the like; fish such as trout, salmon and zander; birds such as geese, ducks, ostriches, and the like.

The isolated cells are optionally genetically modified by gene transfer techniques including 1) transfection by (bio)chemical methods, 2) transfection by physical methods, and 3) virus-mediated transduction. (bio)chemical methods are, for example, calcium phosphate transfection, transfection with DEAE-dextran or lipofection; Physical methods are, for example, electroporation, nucleofection, microinjection, transfection by particle bombardment or transfection by ultrasound; Virus-mediated transduction uses, for example, adenoviruses for short-term infection with high levels of transient expression, herpesviruses for long-term expression or retroviruses for stable integration of DNA into the host cell genome. Cells are expanded after genetic modification.

Isolated cells are incubated with oligonucleotides, eg, before or after genetic modification and/or before or after expansion of the genetically modified cells. Optionally, the isolated cells are purified, eg, by one or more washing steps, before and/or after incubation with the oligonucleotides.

The methods of the present invention optionally include an enrichment step, wherein the isolated cells are enriched via any enrichment method in the art before and/or after incubation with the oligonucleotides. The oligonucleotide is administered back to the isolated cells, for example after a concentration step.

Further, the isolated cells can be, for example, when incubated with an oligonucleotide, before incubation with an oligonucleotide and/or after incubation with an oligonucleotide, after any purification step, after any concentration step, or a combination thereof. are stored frozen Cryopreserved isolated cells, such as immune cells, surprisingly retain the efficacy of antisense oligonucleotide-mediated knockdown of target RNA. The efficacy of cryopreserved isolated cells compared to non-cryopreserved isolated cells in reducing or increasing the transcription and/or translation of the target RNA and/or in reducing the target RNA is very similar.

Isolation according to the present invention involves obtaining cells from a source, for example obtaining immune cells from blood, stem cells from blood of bone marrow or umbilical cord, etc. and/or sub-population of cells from previously isolated cells or cell populations. It means getting a group.

Purification according to the present invention means washing the isolated cells from unwanted substances, such as extracellular substances, for example by means of high pressure liquid chromatography (HPLC), fast protein liquid chromatography (FPLC) or the like.

The method of the present invention optionally comprises an activating step, wherein the isolated cells are subjected to stimulation before and/or after incubation with oligonucleotides, for example using monoclonal antibodies specific for CD3 or CD23 on the surface of T cells by stimulating the cells. or through any activation method in the art by stimulating B cells with CD40 ligand (CD40L). The oligonucleotide is administered back to the isolated cell, for example after an activation step.

The method of the invention optionally comprises an expansion step, wherein the isolated cells are prepared by adding basic fibroblast growth factor (FGF2) to the mesenchymal stem cells, for example before and/or after incubation with the oligonucleotide, or with the oligonucleotide. is augmented by any augmentation method in the art by adding interleukin-2 (IL-2) and/or interleukin-15 (IL-15) to NK cells before and/or after incubation.

The method of the invention optionally comprises a differentiation step, wherein the isolated cells are subjected to interleukin-4 (IL-4) and granulocyte macrophage colony stimulating factor (GM- CSF) are monocytes that differentiate into immature DCs.

The method of the invention optionally comprises a maturation step, wherein the isolated cell is, for example, immature DC, which is added to a toll-like receptor ligand such as for example R848 or LPS or interferon gamma (IFN-γ). ) are natural immature DCs or artificial immature DCs, derived from, for example, the differentiation stage of monocytes as described above, which mature into mature DCs by adding cytokines such as ).

The isolated cells are for example 0 to 21 days, 0 to 20 days, 0 to 19 days, 0 to 18 days, 0 to 17 days, 0 to 16 days, 0 to 15 days, 0 1 to 14 days, 0 to 13 days, 0 to 12 days, 0 to 11 days, 0 to 10 days, 0 to 9 days, 0 to 8 days, 0 to 7 days, 0 days to The oligonucleotide is incubated with the oligonucleotide for a period of 6 days, 0-5 days, 0-4 days, 0-3 days, 0-2 days, or 0-1 days (incubation period). Day 0 is the first day the first oligonucleotide is added to the isolated cells. Oligonucleotides may be added, for example, only once to an isolated cell, or daily for a period or every 2 days, every 3 days, every 4 days, every 5 days, every 6 days, every 7 days, every 8 days, or every 2 days of a period. It is added every 9 days, every 10 days or only on the first and last days of the period, indicating the dosing pattern. Any dosing pattern can be combined during the incubation period, for example the incubation period is from 0 to 9 days, wherein the oligonucleotide is administered daily for 5 days and every 2 days for 4 days. After that period of time, the oligonucleotides are removed, for example, from the isolated cells. Oligonucleotides may be in the nanomolar or micromolar range, for example from 0.1 nmol to 1000 μmol, 0.5 nmol to 900 μmol, 1 nmol to 800 μmol, 50 nmol to 700 μmol, 100 nmol to 600 μmol, 200 nmol to 500 μmol, 300 nmol to 400 μmol, 500 nmol to 300 μmol, 600 nmol to 200 μmol, 700 nmol to 100 μmol or 800 nmol to 50 μmol are added to the isolated cells.

The oligonucleotide reduces expression of the target RNA, for example, from day 0 of the incubation period for at least 10 weeks, for at least 8 weeks, for at least 6 weeks, or for at least 4 weeks. The decrease in expression of the target RNA is independent of, for example, the duration of incubation with the oligonucleotide. These conditions for reducing the expression of the target RNA are reached in each of the incubation periods mentioned above.

The isolated cells are incubated with, for example, one or more, for example 2, 3, 4, 5, 6, 7, 8, 9 or 10 different oligonucleotides. Different oligonucleotides are administered to isolated cells at the same time point during the same time period, at the same time point during a different time period, at different time points during the same time period, or at different time points during a different time period.

Alternatively or additionally, the target RNAs are one or more target RNAs, i.e. the same oligonucleotide reduces expression of more than one target RNA, for example, different oligonucleotides reduce expression of different target RNAs, e.g. have a direct and/or indirect effect on the factor of interest simultaneously or subsequently.

The present invention also relates to an isolated cell, such as an isolated immune cell obtainable by the method of the present invention. The isolated, eg, immune cell, is for use, eg, in a method of preventing and/or treating a disease. Cells are isolated, for example, from a diseased patient or healthy subject, and the isolated cells are incubated ex vivo with an oligonucleotide that hybridizes to a target RNA according to the methods of the present invention. After incubating an isolated cell, such as an isolated immune cell, with an oligonucleotide, the isolated cell is reintroduced into the patient from which the cell was isolated. Alternatively, cells isolated from a healthy subject and incubated ex vivo with an oligonucleotide that hybridizes to a target RNA according to the methods of the present invention are introduced into the patient. A patient suffering from the disease may, for example, increase proliferation, decrease exhaustion, faster cell growth, increase metabolic activity, improve function, improve immunomodulatory effect and increase efficiency and/or increase potency of secretome. can be treated by cells treated with oligonucleotides, such as antisense oligonucleotides that target RNA that directly or indirectly affect Accordingly, the present invention includes allogeneic cell therapy. Oligonucleotides are reintroduced or introduced into the patient, for example, intravenously, intraperitoneally, intramuscularly and/or subcutaneously.

Cells, such as immune cells, for use in a method for preventing and/or treating a disease are isolated immune cells from a patient, healthy subject or a combination thereof, incubated ex vivo with an antisense oligonucleotide that hybridizes with a target RNA according to the present invention. isolated cells such as cells.

The present invention further relates to a pharmaceutical composition comprising the isolated cell of the present invention and a pharmaceutically acceptable excipient. The pharmaceutical composition is for example for use in a method of preventing and/or treating a disease, wherein the pharmaceutical composition is administered, for example intravenously, intraperitoneally, intramuscularly or subcutaneously. Administration of isolated cells or pharmaceutical compositions is based, for example, on infusion or injection.

The disease is, for example, cancer, autoimmune disease, graft-versus-host disease, stroke, spinal cord injury, bone disease, age-related macular degeneration, Parkinson's disease, amyotrophic lateral sclerosis, Alzheimer's disease, diabetes and combinations thereof.

Example

The following examples illustrate different embodiments of the invention, but the invention is not limited to these examples. In the following experiments, no transfection agent is used, ie gymnotic transfer is performed.

Example 1 : Efficacy of knockdown in human T cells without the use of transfection reagents is independent of cell seeding density.

CD3+ human T cells were seeded at different densities per well, i.e. 20.000, 50.000, 75.000 and 100.000 per well in 96-well u-bottom plates on day 0 and 5 μM target-specific antisense oligonucleotide or control oligonucleotide on day 0 and 3 treated with Target protein expression was investigated by flow cytometry on day 6. Robust target knockdown could be achieved irrespective of the initial seeding density. The results are shown in FIG. 1 .

Example 2 : Knockdown persists for several days after removal of antisense oligonucleotides and cryopreservation of cells

CD3+ human T cells were seeded in T25 flasks, activated and treated with mock, control oligo or target-specific antisense oligonucleotides for 6 days. A portion of the cells was then kept in culture (“culture”) and another portion was cryopreserved on day 6. Cryopreserved cells were thawed on day 7 (“freeze/thaw”) and restimulated with conditions “culture” and “freeze/thaw” without addition of control oligos or target-specific antisense oligonucleotides. Target expression was measured over time at the protein level up to 8 days after restimulation. Potential target knockdown was measured at all tested time points and there was no difference between conditions "culture" and "freeze/thaw". Thus, antisense oligonucleotide-mediated knockdown persists throughout the cryopreservation/thaw process as shown in FIG. 2 .

Example 3 : Simultaneous Knockdown of Two Targets in Dendritic Cells

Differentiate CD14+ monocytes into mature dendritic cells (DCs) and target a control oligo, target 1-specific antisense oligonucleotide, target 2-specific antisense oligonucleotide or target 1-specific antisense oligonucleotide and target 2-specific antisense oligonucleotide was treated with a combination of Target protein expression was analyzed on day 3. Robust target knockdown of targets 1 and 2 could be observed in each monotherapy setting. Surprisingly, when cells were treated with a combination of target 1-specific antisense oligonucleotide and target 2-specific antisense oligonucleotide, potent knockdown of both targets could be achieved. The results are shown in FIG. 3 .

Example 4 : Simultaneous knockdown of two targets in T cells

T cells were isolated and activated on day 0 to induce expression of target 3 and target 4. Control oligonucleotide (neg1, final concentration: 5 μM), target 3-specific antisense oligonucleotide (final concentration: 5 μM), target 4-specific antisense oligonucleotide (final concentration: 5 μM), target 3-specific A combination of antisense oligonucleotide and target 4-specific antisense oligonucleotide (final concentration: 5 μM each) or control oligonucleotide (neg1, final concentration: 10 μM) was added to the cells on day 0. Target mRNA and HPRT1 mRNA (housekeeper) expression were analyzed on day 3 by Quantigene SinglePlex assay (ThermoFisher) according to the manufacturer's instructions. Target expression values were normalized to HPRT1 expression values and relative expression compared to mock treated cells was calculated.

The percentage of target protein-expressing cells was examined by flow cytometry on day 5. Relative percentages of target protein-expressing cells compared to mock-treated cells are shown. Robust knockdown of target 3 using target 3-specific antisense oligonucleotides and robust knockdown of target 4 using target 4-specific antisense oligonucleotides were observed. Surprisingly, when target 3 and target 4 antisense oligonucleotides were added to cells in combination, knockdown of both targets was observed with efficacy comparable to the condition in which cells were treated with each antisense oligonucleotide alone. Results are shown in Figure 4a (mRNA) and Figure 4b (protein).

Example 5: Comparison of target 3-specific siRNA and target 3-specific antisense oligonucleotides

T cells were isolated and activated to induce expression of target 3 on day 0. Control oligonucleotides (neg1, final concentration 5 μM) or target 3-specific antisense oligonucleotides (final concentration 5 μM) were added to the cells on day 0 without the use of transfection reagents. As a comparison, standard transfection of untargeted siRNA (Ambion, final concentrations: 30 nM and 60 nM) or target 3-specific siRNA (Ambion, final concentrations: 30 nM and 60 nM) using Lipofectamine 2000 (ThermoFisher) T cells were transfected on day 0 using the protocol. The percentage of target 3 protein-expressing cells was examined by flow cytometry on day 5. Relative percentages of target 3 protein expressing cells compared to mock-treated cells are shown. A strong target 3 knockdown was observed when cells were treated with target 3-specific antisense oligonucleotides. In contrast, there was no reduction in target 3-expressing cells when cells were transfected with target 3-specific siRNA at both test concentrations (30 nM and 60 nM). The results are shown in FIG. 5 .

Claims (15)

dendritic cells, natural killer (NK) cells, peripheral blood mononuclear cells (PBMC), stem cells such as hematopoietic stem cells and/or induced pluripotent stem cells, B cells and combinations thereof in preparation for use in cell therapy. A method of reducing the expression of a target RNA in an isolated immune cell selected from the group consisting of:
incubating an isolated immune cell comprising a target RNA with an antisense oligonucleotide without the use of a transfection means, wherein the antisense oligonucleotide is administered to the isolated cell at least once within a period of 0 to 21 days and the antisense oligonucleotide hybridizes with the target RNA from day 0 to up to 8 weeks of incubation with the antisense oligonucleotide and reduces the transcription of the target RNA or reduces the function of the target RNA or the expression of a protein encoded by the target RNA or a combination thereof. The method of claim 1 , wherein the target RNA is selected from the group consisting of mRNA, pre-mRNA, lncRNA and/or miRNA. The method according to claim 1 or 2, wherein the isolated immune cells are genetically modified by a gene transfer technique before or after incubating the immune cells with antisense oligonucleotides. 4. The method of claim 3, wherein the isolated genetically modified immune cells are expanded before or after incubating the immune cells with antisense oligonucleotides. 5. The method according to any one of claims 1 to 4, further comprising purifying the isolated immune cells before and/or after incubation with the antisense oligonucleotides. 6. The method according to any one of claims 1 to 5, further comprising the step of enriching the isolated immune cells before and/or after incubation with the antisense oligonucleotide, wherein optionally the antisense oligonucleotide is after the enriching step. added to isolated immune cells. 7. The method of any one of claims 1 to 6, wherein when the isolated immune cells are incubated with the antisense oligonucleotides, the immune cells are incubated with the antisense oligonucleotides before the immune cells are incubated with the antisense oligonucleotides. The method further comprising the step of cryopreserving after or in a combination thereof. 8. The method of any one of claims 1 to 7, wherein the target RNA is PD-1, TIGIT, TIM-3, LAG-3, TGFBR, CD39, CD73, 2B4, BTLA, VISTA, CD304, PQR-prot, Chop , XBP1, PERK, FOXP3, GMCSF, IFNg, TNFa, TGFb, IL-1, IL-2, IL-6, IL-10, IL-12, IL-17, IL-9, STAT3, IL-6 receptors and encoding a protein that affects the efficacy and/or safety of immune cells, selected from the group consisting of combinations thereof, or the target RNA is BID, BIM, BAD, NOXA, PUMA, BAX, BAK, BOK, BCL-rambo, BCL -Xs, Hrk, Blk, BMf, a method encoding a protein that affects the proliferation and / or survival of immune cells selected from the group consisting of p53 and combinations thereof. The antisense oligonucleotide according to any one of claims 1 to 8, wherein the antisense oligonucleotide is from 0 to 20 days, 0 to 19 days, 0 to 18 days, 0 to 17 days, 0 to 16 days, 0 days. to 15 days, 0 to 14 days, 0 to 13 days, 0 to 12 days, 0 to 11 days, 0 to 10 days, 0 to 9 days, 0 to 8 days, 0 to 7 days days, 0 to 6, 0 to 5, 0 to 4, 0 to 3, 0 to 2, or 0 to 1 days. 10. The method of any one of claims 1 to 9, wherein the antisense oligonucleotide is administered every day, every 2 days, every 3 days, every 4 days, every 5 days, every 6 days, every 7 days, every 8 days, administered every 9 days or every 10 days. 11. The method of any one of claims 1-10, wherein the antisense oligonucleotides that hybridize with two or more target RNAs are administered at the same time point for the same or different time period or at different time points for the same or different time period. 12. An isolated immune cell for use in a method for the prevention and/or treatment of a disease, wherein the isolated immune cell is derived from a patient or a healthy subject suffering from a disease and according to the method of any one of claims 1 to 11 Incubation ex vivo with an antisense oligonucleotide that hybridizes with a target to reduce expression of the target RNA, and after incubation of the isolated immune cells with the antisense oligonucleotide, the isolated immune cells are reintroduced into the patient or introduced into the patient. immune cells. A pharmaceutical composition comprising cells for use according to claim 12 and a pharmaceutically acceptable excipient. An immune cell according to claim 12 or a pharmaceutical composition according to claim 13 , wherein the patient and/or healthy subject is a human or non-human animal. 15. An immune cell for use according to claim 12 or 14 or a pharmaceutical composition according to claim 13 or 14, wherein the disease is selected from the group consisting of cancer, autoimmune disease, graft versus host disease and combinations thereof. .

Images